G3BP单克隆抗体制备及G3BP在肿瘤组织中表达的检测  

Monoclonal antibody against G3BP: preparation, characterization and its application in analysis of human tumors

作　　者：宁钧宇[1] 由江峰[1] 裴斐[1] 王洁良[1] 崔湘霖[1] 郑杰[1] 

机构地区：[1]北京大学医学部病理学系,100083

出　　处：《中华病理学杂志》2005年第4期215-219,共5页Chinese Journal of Pathology

基　　金：国家自然科学基金资助项目 ( 30170363 );教育部科学技术研究重大项目 ( 01003 );国家重点基础研究发展规划项目( 973 ) ( 2002CB513100 );国家"十五"科技攻关项目(2001BA703B05)

摘　　要：目的　为了研究G3BP(Ras- GAPSH3- bindingprotein)的功能及作用机制,采用原核表达G3BP蛋白诱导免疫,制备抗G3BP单克隆抗体,并检测G3BP在临床常见肿瘤中的表达情况。方法　采用原核表达载体pGEX- 5X1,通过IPTG诱导,使用大肠杆菌BL21,高质量表达GST -G3BP融合蛋白。将纯化后的原核表达蛋白注射BALB/c小鼠诱导产生免疫应答,采用经典杂交瘤技术制备单克隆抗体,并应用酶联免疫吸附试验(ELISA)、蛋白免疫印迹和免疫组织化学染色ABC法进行抗体鉴定和肿瘤检测。结果　筛选出分泌抗G3BP抗体的单克隆杂交瘤细胞,免疫球蛋白亚类鉴定为IgG1,蛋白免疫印迹及抗原竞争抑制实验表明该抗体是特异性抗G3BP抗体。石蜡切片免疫组织化学法检测临床标本,在肺癌、结肠癌、胃癌、乳腺癌等临床肿瘤活检标本中检出G3BP表达明显增高。在乳腺癌临床组织标本中,G3BP表达水平与c -erbB2表达正相关,与淋巴结转移正相关。结论　成功制备了抗人G3BP单克隆抗体,所得到的特异性G3BP单克隆抗体能够应用于ELISA、Western蛋白免疫印迹以及免疫组织化学染色对不同样本的G3BP表达水平进行检测。G3BP在肺癌、结肠癌、胃癌、乳腺癌等肿瘤中表达明显增高。G3BP有可能作为肿瘤标志物辅助对乳腺癌预后的判断。Objective To better understand the molecular mechanism of tumorigenesis and pro gression, the monoclonal antibody against G3BP (Ras-GAP SH3 binding protein), w hich serves as an important downstream effector of Ras signaling, was prepared, characterized and utilized in analysis of various human tumors. Methods By using the prokaryotic expression vector pGEX-5X1, GST-G3BP fusion protein w as expressed in E. coli BL21 under induction of IPTG. Purified GST-G3BP fus ion protein was used to immunize BALB/c mice. The monoclonal antibody against G3 BP was produced through conventional hybridoma method and characterized by ELISA , Western blot and immunohistochemical staining. Results A hybridoma cell line secreting anti-G3BP IgG1 subtype antibody was obtained. Western blot and competitive inhibition assay showed that the anti body was G3BP-specific. Immunohistochemical staining demonstrated that G3BP was over-expressed in formalin-fixed and paraffin-embedded tissues of some human tumors, such as lung cancer, colon cancer, gastric cancer and breast cancer. In breast cancer specimens, the degree of G3BP expression correlated positively wi th the presence of lymph node metastasis and c-erbB2 expression. Conclusions The G3BP-specific monoclonal antibody derived from recombination pro tein can be used in ELISA, Western blot and immunohistochemical assay. It may pr ovide an important tool in analysis of G3BP in in vitro and in vivo expe riments. Besides, G3BP may serve as another prognostic marker for breast cancer.

关 键 词：G3BP 单克隆抗体 制备方法 G3BP 肿瘤组织 基因表达 免疫应答 

分 类 号：R730.4[医药卫生—肿瘤]